Regulation of the cell cycle is essential in all eukaryotes. The E2F transcription factor plays a central role in cell cycle regulation; it also regulates apoptosis and cell type- specific differentiation. The long-term objective of this proposal is to understand the mechanisms by which E2F coordinately regulates these fundamental biological events. The general strategy of the proposal is to study the E2F pathway in Drosophila melanogaster because: 1) the E2F pathway is remarkably conserved in flies and mammals; 2) there are unique opportunities to study cell cycle progression in unperturbed tissue in flies; 3) transgenic flies offer the opportunities to study aspects of the E2F pathway in the intact organism. Three specific aims will be pursued. (1) Identification of the mechanisms underlying ORC1 degradation. We have shown previously that maintaining the proper level of ORC1 is essential for normal cell cycle progression and viability. ORC1 levels rise as a result of E2F-dependent transcription and fall at the end of S phase. We will perform a mutational analysis to identify residues in ORC1 that mediate this degradation, and test whether Cyclin-dependent phosphorylation plays a role in this process. (2) Investigation of E2F-dependent regulation of G2/M progression. Recent experiments suggest that, in addition to its long-appreciated role in regulating G1/S, E2F also regulates the expression of genes involved in controlling G2/M. Using transgenic flies, we will test the role of E2F in regulating the expression of G2/M target genes in vivo. (3) Identification of novel E2F target genes using cDNA microarrays. E2F activity appears to coordinately regulate cell cycle progression, apoptosis, and differentiation, at least in some cell types. However, only a few of the E2F-regulated genes involved in these diverse processes have been identified. We will identify mRNAs that are up-regulated in response to ectopic E2F in imaginal disc cells using cDNA microarrays, with the goal of better understanding how the E2F pathway coordinately regulates proliferation, apoptosis, and terminal differentiation.